Urinary tract infections (UTIs), caused mostly by uropathogenic E. coli (UPEC) affect millions of women each year, with frequent recurrences (rUTI) that do not resolve even with repeated use of antibiotics, causing a significant socio-economic burden. Frequent and long-term prophylactic use of antimicrobials for treatment and prevention of UTIs and other infections has contributed to the evolution of multi-drug-resistance among Gram-negative bacteria. This has become a looming worldwide crisis that has created an urgent need to develop novel treatment and prevention strategies for these infections, including the targeting of bacterial virulence mechanisms. Extracellular fibers termed chaperone-usher pathway (CUP) pili are critical virulence factors in gram negative pathogenic bacteria, with many genomes encoding 10 or more types, all containing dedicated adhesins that promote the recognition of specific receptors and/or drive biofilm formation. FimH, the CUP adhesin joined to the tip of type 1 pili, mediates host-pathogen interactions and bacteria-bacteria interactions critical in biofilm formation and UTI. Chaperone-assisted folding of pilus subunits occurs by a reaction called donor strand complementation (DSC), which templates the folding of subunits into a primed high-energy state. These primed complexes are targeted to the outer membrane usher, a gated channel, which is a molecular machine that catalyzes pilus assembly by driving subunit polymerization in a reaction termed donor strand exchange (DSE). This proposal will pioneer new advances to gain insights into the allostery that governs the interactions needed for CUP pili biogenesis and function. This new direction will provide opportunities for the design of alternative therapeutics to combat the rising problem of multi-drug resistance and Gram-negative bacterial infections in general. This work will elucidate: i) how allosteric modulation of an equilibrium between different conformations of FimH impact host-pathogen interactions (Aim 1); ii) how dynamic allosteric mechanisms of the chaperone and usher facilitate sequential processing of chaperone-subunit complexes and their movement through a molecular machine to promote subunit-subunit polymerization (Aim 2); and; iii) the mechanism of usher activation: how the binding of the initiating chaperone-adhesin complex to the usher results in the reordering of the usher domains to transform chaperone-subunit complexes into a highly stable polymerized fiber extruding across the OM to the bacterial surface (Aim 3). This innovative interdisciplinary research, which is critical for understanding a key step in the virulence of many Gram-negative bacterial pathogens, will use a blend of techniques including bacterial genetics, biochemistry, biophysics, structural biology, cell biology mouse models and high-resolution imaging. Elucidating how a molecular machine converts subunit binding and folding energy into work to assemble highly stable macromolecular CUP fibers and their extrusion to the bacterial surface and the conformational changes governing critical CUP adhesin function will spawn new avenues for drug development.